Abstract Since its discovery from a neurological disease case in California in 1969, enterovirus 71 (EV71) has been found periodically causing epidemics and pandemics worldwide. Since 2008, large outbreaks of EV71 in the Asia-Pacific region have killed thousands of people, mostly infants and young children. EV71 is thought to be the second most important enterovirus after poliovirus, which has been mostly eradicated worldwide. However, the overall research effort devoted to EV71 has been much less than for other major viruses, such as influenza or HIV, for this important virus. As a consequence, very little information was known about how virus replicates and kills host cells, which is one of the major obstacles to develop an effective treatment. A genome-wide unbiased functional screening of the host factors that facilitate virus replication and ensuing cell death will provide step stones to explore deeper into how virus interacts with host factors to replicate and kill host cells. Here we propose to systematically identify EV71 host factors in human cells with a genome-wide knockout screening method based on CRISPR-Cas9 that we have developed independently. In preliminary studies, we have shown that our method can identify viral host factors with high sensitivity and specificity and, more importantly, strong phenotypes. Meanwhile, we will also change screening strategy so the host genes with weaker phenotype will also be identified. Thus, we expect to identify EV71 host factors systematically without bias to gain the comprehensive human host factor?virus interaction map, which will shed light on how human host factors facilitate EV71 replication and/or host cell death. In addition, the human host factors identified will provide potential therapeutic targets for treatment of EV71 infection, which is an ongoing public health problem without specific treatment available.